1. Field of the Invention
The present invention is in the field of synthesis methods for the preparation of commercial cephalosporin antibiotics, of which there are presently a significant number, these therapeutic agents now being in their fourth generation. The large variety of side chains to be found in commercial cephalosporins and the significant economic importance of the cephalosporins has placed increased importance on achieving more economic and efficient methods of preparing key intermediates which permit ready synthesis of the various cephalosporins.
One of these key intermediates is 7-amino-cephalosporanic acid (7-ACA), which may be represented by the following formula: ##STR1## Currently, 7-ACA is produced from Cephalosporin C. Cephalosporin C itself is a fermentation product which is the starting point for nearly all currently marketed cephalosporins. However, synthetic manipulation to produce these various commercial cephalosporins basically starts in most cases with the 7-aminocephalosporanic acid, which must be derived from the Cephalosporin C by cleavage of the 7-aminoadipoyl side chain. Typical commercial cephalosporins derived synthetically from 7-ACA, and which thus have the 3-acetyloxymethylene side chain, include cefotaxime, cephaloglycin, cephalothin, and cephapirin.
Another of the key intermediates is 7-aminodeacetylcephalosporanic acid (7-ADAC), which may be represented by the following formula: ##STR2## Currently, 7-ADAC is also produced from Cephalosporin C by removal of the 7-D-.alpha.-aminoadipoyl side chain, together with conversion of the 3-acetyloxymethyleneside chain to 3-hydroxymethyl. 7-ADAC is a useful intermediate compound in the synthesis of cephalosporins containing modified substituents at the C-3 position.
Currently, the method of choice in the art for cleaving the 7-aminoadipoyl side chain is chemical. The basic imino-halide process requires blocking of the amino and carboxyl groups on the(7-aminoadipoyl side chain, and several methods for accomplishing this are currently used. However, as presently employed, the chemical cleavage process has serious disadvantages. Among these are the requirements of a multi-step and complex process, extremely low operating temperatures, expensive reagents, significant quantities of process by-products resulting in effluent treatment problems, and purification of a highly impure starting material before chemical treatment begins. Consequently, there has been an ongoing search for a microbiological or fermentative process which would achieve enzymatic deacylation of Cephalosporin C to provide 7-aminocephalosporanic acid on a more economic basis than the chemical process currently in use.
However, this search for a successful microbiological process has largely proved futile. This is a result, as is made clear in the literature, of the structure, and especially the stereochemistry, of the-aminoadipoyl side chain of the Cephalosporin C molecule, since penicillin has been successfully deacylated by enzymatic cleavage using penicillin acylase produced by a variety of microorganisms. Reports of successful one-step enzymatic deacylation of Cephalosporin C in the literature, on the otherhand, are often unreproducible or provide only very marginal yields.
Accordingly, the present invention is particularly in the field of preparing the key cephalosporin intermediate 7-ACA, and more particularly, in the field of bioprocesses for the preparation of 7-ACA.
To date, the search for a successful bioprocess for making 7-ACA has largely proved futile, certainly with respect to one of commercial scale. For example, while it has been possible to prepare 6-amino penicillanic acid (6-APA) by direct fermentation and/or by enzymatic treatment of penicillin G, leaving only ring expansion necessary to give 7-ADCA, it has been found that, unfortunately, the Cephalosporium or Streptomyces enzymes which carry out ring expansion in the normal metabolic pathways of these microorganisms do not accept 6-APA as a substrate. These enzymes, which are collectively referred to in the art as the DAOCS or expandase enzyme, are defined As enzymes which catalyze the expansion of penam ring structures found in penicillin-type molecules to ceph-3-em rings, as found in the cephalosporins. Hereafter, these enzymes will be referred to collectively as "the expandase enzyme".
A substrate on which the expandase enzyme does operate is penicillin N, which upon ring expansion and hydroxylation, gives deacetylcephalosporanic acid (DAC). Here, it is only necessary to cleave the (D)-.alpha.-aminoadipoyl side chain to give 7-ADAC, but this side chain has proven stubbornly resistant to enzymatic cleavage, giving only unacceptably low yields.
In accordance with the present invention it has been possible to achieve an efficient bioprocess wherein a penicillin compound (having an adipoyl side chain) is produced by a novel fermentation process in high titers, said penicillin compound being an acceptable substrate for the expandase enzyme which is produced in situ by the same microorganism which produces the penicillin compound, having been transformed to express said expandase enzyme. The expandase enzyme then operates to ring expand the penicillin compound to a cephalosporin compound in high yields.
The adipoyl-7-ADCA produced by in situ action of the expandase enzyme has a 3-methyl (--CH.sub.3) side lo chain, whereas 7-ACA, the final product, has a 3-acetyloxymethyl [--CH.sub.2 OC(O)CH.sub.3 ] side chain. In order to convert the 3-methyl to a 3-acetyloxymethyl side chain, in accordance with the present invention there is also expressed in situ two further enzyme activities in addition to the expandase activity. These are, in order, an hydroxylase and an acetyltransferase, and both are the expression products of genes with which the microorganism producing the penicillin compound has also been transformed. The hydroxylase enzyme converts the 3-methyl side chain of adipoyl-7-ADCA to 3-hydroxymethyl, and the acetyltransferase enzyme converts this 3-hydroxymethyl side chain to the 3-acetyloxymethyl side chain of 7-ACA.
And, importantly in the last critical step of the method of the present invention, the side chain of the penicillin compound, now a cephalosporin compound, is removable by another enzyme system in surprisingly high yields. The unexpected result of this unique, total bioprocess which comprises the present invention, is the production of 7-ACA in surprisingly high yields, and with sufficient economy to represent a reasonable alternative to currently used methods of chemical and biochemical processing.
2. Brief Description of the Prior Art
The novel bioprocess of the present invention provides a unique and surprisingly efficient method for preparing 7-ACA as an economically viable alternative to current chemical synthesis. Continuing efforts in the art to devise such a bioprocess have experienced repeated failure. For example, EP-A-0 422 790 discloses DNA encoding isopenicillin N:acyl-CoA acyltransferase activity of Aspergillus nidulans and its use in generating useful cephalosporins in penicillin-producing fungi, which has not heretofore been accomplished in the art. But, this is described as being by way of disruption or displacement of the acyltransferase gene along with addition of genes encoding the epimerase and expandase enzymes from cephalosporin-producing organisms; moreover, no useful transformation and expression result is actually achieved, apparently. Furthermore, had transformation been successful, it still would not have been useful for the purposes of the present invention, since the problem of how to remove the D-.alpha.-aminoadipoyl side chain would still remain. Such a failed attempt in the art to obtain significant results in producing commercial cephalosporin intermediates from penicillin-producing fungi cultures is in complete contrast to the results achieved with the method of the present invention.
The first enzymatic bioprocess step in the method of the present invention is ring expansion of adipoyl-6-APA, carried out by an expandase enzyme which is the expression product of an expandase gene with which the non-recombinant P. chryaogenum host as been transformed. The use of synch an expandase enzyme has been explored in the prior art. For example, Cantwell et al., in Curr Genet (1990) 17:213-221, have proposed a bioprocess for preparing 7-ADCA by ring expansion of penicillin V followed by enzymatic hydrolysis of the resulting deacetoxycephalosporin V to form 7-ADCA. This proposal is based on the availability of a cloned penicillin N expandase gene (cefE) from S. clavuligerus: Kovacevic et al., J. Bacteriol. (1989) 171:754-760; and Ingolia et al. U.S. Pat. No. 5,070,020. However, since the expandase operates on penicillin N, its natural substrate, but not on penicillin V, the proposal requires genetic engineering to produce a modified expandase gene which can ring-expand the penicillin V. The required modification was not achieved by Cantwell et al., however, and they only succeeded in transforming Penicillium chrysogenum with the cef E gene from Streptomyces clavuligerus and getting low-level expression of the DAOCS (expandase) enzyme.
The expandase enzyme has been well studied in the art, both with respect to its activity and its genetic sequence. For example, in Wolfe U.S. Pat. Nos. 4,510,246 and 4,536,476, cyclase, epimerase and ring expansion enzymes were isolated separately from a cell free extract of prokaryotic .beta.-lactam producing organisms, including Streptomyces clavuligerus, to provide stable enzyme reagents. Dotzlaf U.S. Pat. No. 5,082,772 (EP-A-0 366 354) describes an isolated and purified expandase enzyme from S. clavuligerus which is characterized, including by a terminal residue and amino acid composition, and is said to have a molecular weight of about 34,600 Daltons. This is in contrast, however, to the molecular weight of 29,000 assigned to what would appear to be the same enzyme in U.S. Pat. No. 4,536,476. EP-A-0 233 715 discloses isolation and endonuclease restriction map characterization of the expandase gene obtained from S. clavuligerus and expression of recombinant expandase-encoding DNA (yielding active expandase enzyme) in an S. clavuligerus strain lacking the capability of cephalosporin production. Ingolia et al. U.S. Pat. No. 5,070,020 (EP-A-0 341 892) discloses the DNA sequence encoding the expandase enzyme obtained from S. clavuligerus and describes the transformation of a P. chrysogenum strain with an expression vector containing said DNA sequence, thereby obtaining expression of the expandase enzyme. While it is suggested that this enzyme is useful for the expansion of substrates other than penicillin N, there is no actual demonstration of such an expansion.
The work described above has focused on the expandase enzyme derived from prokaryotic S. clavuligerus. An enzyme apparently having the same ring expansion activity is also expressed by strains of eukaryotic Cephalosporium acremonium (also referred to as Acremonium chrysogenum). However, in such strains expandase activity is expressed by a bifunctional gene (cefEF), which also expresses the DACS (hydroxylase) activity whose natural function is to convert the desacetoxycephalosporanic acid (DAOC) product of the expandase enzyme to deacetyl cephalosporin C (DAC). The result of this expression is a single, but bifunctional expandase/hydroxylase enzyme. While there have been efforts to separate the activities of these two gene products, none have yet been successful. For example, EP-A-0 281 391 discloses the isolation and DNA sequence identification of the DAOCS/DAOS gene obtained from C. acremonium ATCC 11550 together with the corresponding amino acid sequence of the enzyme. A Penicillium is transformed and expresses the enzymes, however, the attempted conversion of penicillins G and V to the corresponding cephalosporins is never demonstrated. Further, despite a suggestion that genetic engineering techniques provide a ready means to separate the genetic information encoding DAOCS from DAGS and separately express them, no actual demonstration of such separation is set forth.
The DAOCS/DACS (expandase/hydroxylase) enzyme of C. acremonium has also been well studied in the art, both with respect to its activity and its characteristics and genetic sequence. For example, in Demain U.S. Pat. Nos. 4,178,210; 4,248,966; and 4,307,192 various penicillin-type starting materials are treated with a cell-free extract of C. acremonium which epimerizes and expands the ring to give a cephalosporin antibiotic product. Wu-Kuang Yeh U.S. Pat. No. 4,753,881 describes the C. acremonium enzyme in terms of its isoelectric point, molecular weights, amino acid residues, ratio of hydroxylase to expandase activities and peptide fragments.
The acetyltransferase enzyme of C. acremonium has also been described in the art, with respect to its activity, characteristics, restriction mapping, and nucleotide and amino acid sequences. For example, see EP-A-0 437 378 and EP-A-0 450 758.
The prior art discussed above deals with only a single aspect of the present invention, i.e., the transformation of a P. chrysogenum strain with genes expressing the expandase and expandase/hydroxylase enzymes and obtaining expression of those enzymes. The art, however, has only used the expressed enzymes to ring-expand penicillin N, not penicillins G and V. Even in that case, penicillin N has a 7-position side chain which cannot be cleaved enzymatically to leave a free amino group. The present invention is based on the surprising discovery that an adipoyl side chain can be efficiently added by a P. chrysogenum strain, that the expandase enzyme expressed in situ can use that compound efficiently as a substrate for ring expansion to adipoyl 7-ADCA, that hydroxylase and acetyltransferase enzymes also expressed La situ can use the adipoyl-7-ADCA as a substrate to produce the 3-acetoxymethyl side chain of 7-ACA, and that the adipoyl side chain can then be efficiently removed by yet another enzyme to give 7-ACA. While various isolated fragments of the present invention may be found in the prior art, there has been no suggestion that they be combined to give the unexpected results obtained with the method of the present invention.
For example, production of 6-adipoyl penicillanic acid is known in the art; see Ballio, A. et al., Nature (1960) 185, 97-99. The enzymatic expansion of 6-adipoyl penicillanic acid, but only on an in vitro basis, is also known in the art; see Baldwin et al., Tetrahedron (1987) 43, 3009-3014; and EP-A-0 268 343. And, enzymatic cleavage of adipoyl side chains is also known in the art; see for example, Matsuda et al., J. Bact. (1987) 169, 5815-5820.
The adipoyl side chain has the following structure: COOH--(CH.sub.2).sub.4 --CO--, while two side chains of closely related structure are those of glutaryl, having the following formula: COOH--(CH.sub.2).sub.3 --CO--, and of (D)-.alpha.-aminoadipoyl, having the formula: COOH--CH(NH.sub.2)--(CH.sub.2).sub.3 --CO--. The enzymatic cleavage of glutaryl side chains is known in the art. See, e.g., Shibuya et al., Agric. Biol. Chem. (1981) 45, 1561-1567, and U.S. Pat. No. 3,960,662; Matsuda and Komatsu, J. Bact. (1985) 163, 1222-1228; Matsuda et al., J. Bact. (1987) 169, 5815-5820; Jap. 53-086084 (1978--Banyu Pharmaceutical Co. Ltd.); and Jap. 52-128293 (1977--Banyu Pharmaceutical Co. Ltd.). Also, EPA-A-0 453 048 describes methods for improving the adipoyl-cleaving activity of the glutaryl acylase produced by Pseudomonas SY-77-1. By substituting different amino acids at certain locations within the alpha-subunit, a three to five times higher rate of adipoyl cleavage (from adipoyl-serine) was observed. It should be noted that although FP-A-0 453 048, apparently, demonstrates an acylase with improved activity towards adipoyl-side chains, it does not describe any ways (either chemical or through a bioprocess in any way analogous to that described in the instant specification) in which an adipoyl-cephalosporin might be generated in the first place.
Where a (D)-.alpha.-aminoadipoyl side chain is present, it is known in the art to first enzymatically remove the amino group and shorten the side chain with a (D)-amino acid oxidase, leaving a glutaryl (GL-7) side chain, with removal of the glutaryl side chain by a second enzyme (glutaryl acylase). Such a two-step cleavage is disclosed in Matsuda U.S. Pat. No. 3,960,662; EP-A-0 275 901; Jap. 61-218057 (1988--Komatsu, Asahi Chemical Industry Co.); WO 90/12110 (1990--Wong, Biopure Corp.); and EP-A-0 436 355, Isogai et al., also Bio/Technology (1991) 9, 188-191.
It is also known in the art to carry out one-step cleavage of the (D)-.alpha.-aminoadipoyl side chain, particularly using recombinant techniques. See, e.g.:
One-step (D)-.alpha.-aminoadipoyl side chain cleavage:
Jap. 53-94093 (Meiji, Pseudomonas sp. BN-188); PA0 Jap. 52-143289 (=U.S. Pat. No. 4,141,790, Meiji, Aspergillus sp.); PA0 U.S. Pat. No. 4,774,179 (Asahi 1988, Pseudomonas sp. SE-83 and SE-495), =Jap. 61-21097 and Jap. 61-152286; PA0 Fr. Pat. 2,241,557 (Aries 1975, Bacillus cereus var. fluorescens); PA0 Jap. 52-082791 (Toyo Jozo 1977, Bacillus megaterium NRRL B 5385); PA0 EP-A-0 321 849 (Hoechst, Pseudomonas, Bacillus subtilis, .gamma.-glutamyl transpeptidase); PA0 EP-A-0 283 218 and U.S. Pat. No. 4,981,789 (Merck, Arthrobacter viscosus); PA0 Jap. 60-110292 (Asahi 1985, Comamonas, recombinant E. coli with gene from Comamonas sp. SY-77-1, one-step conversion); PA0 Jap. 61-152-286 (Asahi 1986, Pseudomonas, recombinant E. coli with gene from Pseudomonas sp. SE83, genetic sequences described and claimed, one step process already claimed in U.S. Pat. No. 4,774,179); PA0 Jap. 63-74488 (Asahi 1988, Trigonopsis variabilis, Comamonas, recombinant E. coli expression of D-amino acid oxidase and GL-7-ACA acylase construct). PA0 EP-A-0 475 652 (Fujisawa, cephalosporin C acylase and its production via recombinant technology).
EP-A-0 322 032, EP-A-0 405 846, and U.S. Pat. No. 5,104,800 (Merck, Bacillus megaterium); PA1 1) maintaining in a culture medium capable of sustaining its growth, a strain of Penicillium chrysogenum which produces isopenicillin N and adding to said culture medium an adipate feedstock comprising any one or more of adipic acid, or its salts and esters which are capable of being assimilated and utilized by said strain of Penicillium chrysogenum to produce adipoyl-6-aminopenicillanic acid (adipoyl-6-APA), whereby said adipoyl-6-APA is produced; PA1 2) carrying out the following enzymatic conversions by in situ expression of the corresponding gene: PA1 3) contacting said adipoyl-7-ADAC with an adipoyl amidase whereby the adipoyl side chain is removed and the 7-ADAC product is formed; and said product is then isolated. PA1 1) maintaining in a culture medium capable of sustaining its growth, a strain of Penicillium chrysogenum which produces isopenicillin N and adding to said culture medium an adipate feedstock comprising any one or more of adipic acid, or its salts and esters which are capable of being assimilated and utilized by said strain of Penicillium chrysogenum to produce. adipoyl-6-aminopenicillanic acid (adipoyl-6-APA), whereby said adipoyl-6-APA is produced; PA1 2) carrying out the following enzymatic conversions by in situ expression of the corresponding gene: PA1 3) contacting said adipoyl-7-ACA with an adipoyl amidase whereby the adipoyl side chain is removed and the 7-ACA product is formed; and said product is then isolated. PA1 "7-ACA" means 3-[(acetoyloxy)-methyl]-7-amino-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene- 2-carboxylic acid; PA1 "adipoyl-6-APA" means [2S-(2.alpha., 5.alpha., 6.beta.)]-3,3-dimethyl-7-oxo-6-[hexane-1,6-dioyl) amino]-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid; PA1 "adipoyl-7-ADCA" means 3-methyl-7-[(hexane-1,6-dioyl)amino]-3-methyl-8-oxo-5-thia-1-azabicyclo[4. 2.0]oct-2-ene-2-carboxylic acid; and PA1 "adipoyl-7-ADAC" means 3-hydroxymethyl-7-[(hexane-1,6-dioyl)amino]-3-methyl-8-oxo-5-thia-1-azabic yclo [4.2.0]oct-2-ene-2-carboxylic acid. PA1 Cephalosporium acremonium PA1 Cephalosporium sp. PA1 Emericellopsis PA1 Paecilomyces PA1 Scopulariopsis PA1 Diheterospora PA1 Spiroidium PA1 Anoxiopsis PA1 Streptomyces clavuligerus PA1 S. lipmanii PA1 S. wadayamensis PA1 S. todorominensis PA1 S. filipinensis cephamycini PA1 S. heteromorphus PA1 S. panayensis PA1 S. griseus PA1 S. cattleya PA1 Nocardia lactamdurans PA1 Flavobacterium sp. PA1 Alcaligenes denitrificans PA1 Mycoplana bullata PA1 Providencia rettgeri PA1 Lysobacter lactamgenus
One-Step-Recombinant: Ceph C.fwdarw.7-ACA:
Various aspects of methods for producing 7-ADAC are known in the art. For example, see U.S. Pat. Nos. 3,304,236 and 3,972,774 (Eli Lilly & Co.); EP-A-0 454 478 (Shionogi & Co., Ltd.); and published Japanese application 04 53,499 (Shionogi & Co., Ltd.).
Reference to Copending Application
Reference is made to copending application Ser. No. 07/933,469, filed Aug. 28, 1992 (Attorney Docket No. 18532IA), which discloses a bioprocess for making 7-ADCA that relies on expression of the activity of the expandase enzyme in a P. chrysogenum transformant in the same manner as the bioprocess for making 7-ADAC and 7-ACA described herein. However, in the present bioprocess, additional transformations are utilized for the expression of additional enzymatic activities, in order to achieve a wholly different recombinant metabolic pathway to distinct final products, none of which is suggested in the copending application.
In order to facilitate a better understanding of the method of the present invention and the teachings of the prior art references discussed above, set out immediately below is a representation of the various stages in the metabolic pathways leading to adipoyl-6-APA, adipoyl-7-ADCA, adipoyl-7-ACA, and 7-ACA, the intermediate products, and the enzymes which carry out the transformations involved. ##STR3##